The invention relates to the deposit upon substrates of small quantities of fluid in a precise manner and in arrays of desired density and consistency. The invention is useful, for instance, in carrying out reactions, in providing accurate overlays of deposits, and, in particular, in preparing microscope slides and membranes with biological materials.
The invention also relates to array products produced by the novel deposit techniques and to methods of analysis that employ the deposit techniques.
In the field of biochemistry it is desirable to accurately and efficiently deposit tens, hundreds, thousands and tens of thousands of samples of differing compositions on reaction or examination areas. Improvement in the speed of deposition, the precision of the size, shape, quantity and location of the deposits and the control over density of the deposits can lead to important advantages.
In particular, well developed biological analytical technology, and recently developed xe2x80x9cLab on a Chipxe2x80x9d or xe2x80x9cGene Chipxe2x80x9d techniques require creation of dense arrays of fluorescently labeled micro-organisms and DNA assays in a two dimensional field. It is desirable to place the arrays on a conventional microscope slide, and to create many such slides simultaneously in a manufacturing process.
In important applications, single stranded DNA or PNA or other biological elements in the form of fragments carrying known information are distributed onto the surface of a planar field array containing up to possibly 100,000 objects per microscope slide. The objects of the array represent discriminating sequence information. Different laboratories have targeted the objects of the array to have various spot sizes over a range of the order of 25 to 250 xcexcm in diameter, depending primarily upon the total number of objects anticipated in the array. The objects of the array are probed with fluorescently labeled fragments of potential complementariTy. When a match occurs between these fragments and hybridization occurs, a positive is scored by observing fluorescence at the site of hybridization. By manipulating the deposition of complementary strands or fragments into the array and scoring xe2x80x9chitsxe2x80x9d, many levels of information can be inferred.
For gene chip technology to proceed to complete fruition, as well as to improve the application of previous analytical techniques, economical instruments have been needed that can rapidly and accurately create the dense array of objects over a large field portion of a glass microscope slide or slide-like member that occupies an area approximately 22 mm wide and 50 mm long of a slide that is nominally 25 mmxc3x9775 mm.
In the deposition upon a microscope slide of discrete, minute quantities of a large variety of fluid materials, the volume deposited at a discrete spot typically may be from a few pico liter to a fraction of a micro liter, depending upon the application. The biological material carried in this fluid can range from a few strands of short oligonucleotides in a water solution to a high concentration of long strands of complex proteins. The properties of these fluids vary enormously. Some are akin to water while others are far more viscous, resembling a light oil or honey. The range of fluids that may be employed also exhibits wide differences in evaporative characteristics and in other properties.
Such large range of property variations in fluids of interest has caused great difficulties for any single type of process to operate over a wide range.
Certain processes employing photolithographic techniques have offered excellent positional accuracy of the objects and high dot density but have great limitations due to cost and due to the limited range of biological and chemical techniques and substrates that are applicable. These techniques typically construct short segments of DNA or other molecules by adding single bases, one at a time.
Certain other processes for forming arrays of dots of biological material have utilized piezo micro cylinders to aspirate and jet small volumes of fluid containing the material while others have used processes akin to a fountain pen, comprising a xe2x80x9cquillxe2x80x9d deposition tool. An assemblage of quills suck up a desired amount of fluid and by tapping a quill upon the receiving substrate, the meniscus holding the fluid in the gap of the quill breaks, due to inertia of the fluid within the suddenly stopped tool, so that a drop of fluid is effectively propelled from inside the quill to the impacted surface.
The development of such techniques has occurred against the background of the quite old technique for forming much larger deposits, of transferring a portion of fluid by a pin or a set of pins that are e.g. dipped in a fixed reservoir containing fluid to be transferred and moving the pins into position to contact a usually soft substrate to form relatively large spots. Some of these instruments are known as xe2x80x9creplicatorsxe2x80x9d. An example of a product produced by such prior pins would be a 22 cmxc3x9722 cm bioassay plate carrying 0.6 mm diameter spots located on a grid 1 mm on center. This spot density is approximately 3 orders of magnitude too low from that needed for current xe2x80x9cGene Chipxe2x80x9d applications, and the previously known techniques are impractical for present purposes for a number of other reasons as well.
One purpose of the invention is to provide a technology adapted to the deposition of very small drops of fluids, e.g. drops that form spots of less than about 375 or 300 xcexcm diameter, and in important cases much smaller than that, and at correspondingly high densities (as used in this application, the term fluid xe2x80x9cdropxe2x80x9d refers to a very small quantity of fluid, and not to any particular shape of the fluid volume). The fluids and the resultant dots permissibly exhibit a wide range of properties such as viscosity, evaporative characteristics, surface tension, wettability, surfactant characteristic, dynamic contact angle and free surface energy. These and numerous other objectives are achieved by a number of broad features and preferred embodiments which are individually novel and important and which in many cases cooperate to achieve highly effective results.
According to one aspect of the invention, an apparatus for depositing fluid dots on a receiving surface in an array suitable e.g., for microscopic analysis reaction and the like, is provided, comprising a deposit device and a fluid source which are cooperatively related to enable the deposit device to precisely size a drop of fluid of small diameter on a drop-carrying surface of the device, transport mechanism for positioning the device at a precisely referenced lateral position over the receiving surface and drive mechanism for moving the deposit device, relatively, in deposition motion toward and away from the surface, the apparatus adapted, by repeated action, to deposit the drops of fluid precisely in a desired array, preferably the apparatus being computer controlled.
Preferred embodiments have one or more of the following features.
The drop-carrying surface has a diameter less than 375 micron, preferably less than 300 micron, preferably between about 15 or 50 micron and 250 micron.
The drop-carrying surface is bound by a sharp rim that defines the perimeter of the drop of fluid.
The deposit device is a pin or pin-like structure having an end surface that carries the fluid drop, preferably the pin or pin-like structure having sides that intersect with the end surface to define a sharp peripheral drop-defining rim.
Another broad aspect of the invention is an apparatus for depositing fluid drops on a receiving surface per se, comprising a deposit device and a fluid source which are cooperatively related to provide to a drop-carrying surface of the deposit device a precisely sized drop of fluid, the deposit device being a pin or pin-like structure having an end surface that serves as the drop-carrying surface, the pin or pin-like structure having sides that intersect with the end surface to define a sharp peripheral drop-defining rim.
Preferred pins or pin-like structures have an end surface that is generally flat and side surfaces that are cylindrical and smooth.
In preferred cases the deposit device is mounted for compliance in the direction of the deposition motion when the deposit device engages the receiving surface, preferably the deposit device being compliantly displaceable by overcoming resistance of a resilient member or weight, preferably, when the deposit device is an axially slidable pin or pin-like structure, the means for urging comprises a coaxial spring or a weight acting on the pin or pin-like structure. Also preferably, the drive mechanism for the deposit device is constructed to overtravel beyond a level at which the compliantly displaceable deposition device engages the receiving surface.
In important cases the deposit device, at the time of deposit, is laterally constrained to a reference position, as by the deposit device being mounted on a flexure system that defines the referenced lateral position of the deposit device, or the deposit device is mounted in a manner permitting its displacement relative to its mounting upon its engagement with the receiving surface, at the time of engagement of the device with the receiving surface, the deposit device being subjected to a lateral force or turning moment that engages the deposit device with at least one lateral reference surface, preferably the deposit device being a pin or pin-like structure which is free to slide axially relative to its mounting upon engagement of the pin or pin-like structure with the receiving surface, and which is urged against the lateral reference surface by a spring, a weight such as the weight of the device, an eccentric weight, or the device being tilted, or by electrical or magnetic forces acting upon the pin or pin-like structure, to produce a lateral force or moment toward the reference surface.
In many important cases, the fluid source is a mobile fluid storage device that is movable relative to an array of deposit locations, the fluid storage device being constructed and arranged to resupply the deposit device at various locations along the array.
Another broad aspect of the invention is an apparatus for depositing fluid drops on a receiving surface, comprising a deposit device and a fluid source which are cooperatively related to provide to the deposit device a drop of fluid, transport mechanism for positioning the deposit device over a receiving surface and drive mechanism for moving the deposit device, relatively, in deposition motion toward and away from the receiving surface, the apparatus adapted, by repeated action, to deposit the drops of fluid in a desired array, the fluid source being a mobile fluid storage device that is movable relative to the array of deposit locations, the fluid storage device being constructed and arranged to resupply the deposit device at various locations along the array.
In preferred embodiments employing a mobile storage device, the deposit device and the mobile storage device are constructed to supply drops to the deposit device in the immediate vicinity of the deposit locations for respective drops, preferably the mobile fluid storage device and the deposit device being coupled for transverse motion relative to the array and decoupled for movement of the deposit device toward and away from the receiving surface.
In many cases the mobile storage devices are preferably constructed and arranged to be replenished from a remotely located large reservoir.
In many cases a mobile storage device holds a volume of fluid having a free surface into which the deposit device is lowered and raised to obtain a fluid drop, preferably the mobile storage device being constructed to store a multiplicity of isolated fluid volumes in the wells of a multiwell plate, the apparatus constructed to obtain its fluid from a selected volume of the plate.
In other important cases a mobile storage device defines a generally annular fluid retention surface or ring (the term xe2x80x9cannularxe2x80x9d or xe2x80x9cringxe2x80x9d being used to refer broadly to a member that has opposed or adjacent, surfaces that can hold a mass of fluid between them by surface tension effects, accessible to a deposit device), and the deposit device is constructed to move within the annular retention surface from retracted to extended positions, in the retracted position the drop-carrying surface of the deposit device being retracted from the surface of fluid retained by the annular surface of the storage device, and in the extended position the drop-carrying surface of the deposit device being projected through and beyond the surface of the retained fluid.
Another broad aspect of the invention is an apparatus for depositing fluid drops-on a receiving surface in an array suitable for microscopic analysis, comprising a deposit device and a fluid source which are cooperatively related to provide to a drop-carrying surface of the deposit device a precisely sized drop, and a drive mechanism for moving the deposit device, relatively, in deposition motion toward and away from the receiving surface, the storage device defining a generally annular fluid retention surface, and the deposit device being constructed to move within the annular retention surface from retracted to extended positions, in the retracted position the drop-carrying surface of the deposit device being retracted from the surface of the fluid retained by the annular surface of the storage device, and in the extended position the drop-carrying surface of the deposit device being projected through and beyond the surface of the retained fluid.
In preferred embodiments, a member that defines an annular fluid retention surface is associated with a driver that moves the member relative to the deposit device to a replenishment volume in which the member is immersed to receive a supply of fluid.
In certain preferred embodiments the deposit device is a pin or pin-like structure e.g. having one or more of the features described above, the pin or pin-like structure being mounted within the confines of an annular fluid retention surface and arranged to move axially relative thereto.
Preferably, fluid retaining surfaces of the annular storage device have a hydrophilic surface, e.g. a surface roughness of at least 1000 microinch or a surface energy greater that about 2500 mN/m, preferably the surface comprising tungsten, and preferably, e.g. when cooperating with the annular member to pick up a supply of fluid the drop-carrying surface or tip of the deposit device has a surface of surface energy greater than about 2500 mN/m, preferably the surface comprising tungsten.
The apparatus of any of the aspects and preferred embodiments described preferably include a cleaning system and a control system adapted to control relative movement of the deposit device to a depositing relationship to the receiving surface and a cleaning relationship to the cleaning system.
Another broad aspect of the invention is an apparatus for depositing an array of dots on a receiving surface, comprising a deposit device in the form of a pin or pin-like structure having an end surface capable of precisely defining a small drop of fluid, a source of fluid for the deposit device, mechanism for moving the deposit device relatively over an array of spaced apart deposit locations of a receiving surface, mechanism for repeatedly moving the deposit device, relatively toward and away from the receiving surface to deposit respective drops of fluid at selected deposit locations, a cleaning system, and a control system adapted to control relative movement of the deposit device between a resupply relationship to the source, a depositing relationship to the substrate and a cleaning relationship to the cleaning system.
In embodiments in which the deposit device is associated with a mobile supply device that travels with it, the deposit device and mobile supply device are preferably movable together to the cleaning system in response to the control system, preferably the mobile supply device being an annular member through which the deposit device operates. Preferably the cleaning system has a nozzle for directing a flow of air past the annular structure, preferably a cleaning or drying station comprising a circular nozzle constructed to discharge a conical flow of fluid, preferably compressed air, high pressure liquid, an aerosol or heated air against a deposit device or mobile fluid source, preferably the deposit device being a pin or pin-like structure surrounded by a mobile reservoir in the form of an annular member capable of holding a supply of fluid by surface tension effects, the nozzle flows directed to dislodge retained fluid, to clean or to dry the respective parts; in some uses preferably an circular storage device is associated with a heater, e.g., an induction heater.
In certain preferred embodiments of the various aspects and features described, there are provided a set of at least two of the deposit devices, at least one fluid source for providing a drop of fluid on each deposit device, and mechanism for moving the pins together transversely over an array of spaced apart deposit locations of the receiving surface, preferably there being at least four of the deposit devices comprising a deposit head. Preferably the apparatus includes mechanism for repeatedly moving each deposit device independently, or mechanism for moving each deposit device simultaneously, relatively, toward and away from the receiving surface to deposit respective drops at respective deposit locations on the receiving surface.
For simultaneous actuation, preferably two or more deposit devices are mounted on a common support, driven by a common driver to deposit respective fluid drops on the receiving surface. In cases in which each deposit device is associated with a respective storage ring, the storage rings are also mounted on a common support, driven by a common drive; preferably the spacing of the rings corresponds to the spacing of a multiwell storage plate into which the rings are immersed for resupply. In cases in which the deposit device is lowered directly into fluid and raised to obtain its drop, preferably the spacing of the deposit devices corresponds to the spacing of wells of a predetermined multiwell plate, the multiwell plate being a mobile fluid supply that is constructed to accompany the deposit device across the substrate. In the case of supply rings or direct dipping of the deposit devices, preferably in the spacing corresponds to well-to-well spacing of wells of a 96, 384, 864 or 1536 well plate, or a spacing of 9 mm or a submultiple of 9 mm.
The various apparatus described preferably have one or more of the following features.
The deposit device and its mounting limits the force of engagement of the deposit device upon the receiving surface to less than 1 gram, preferably less than 0.5 gram, preferably to about 0.3 gram.
The deposit device has a natural frequency greater than 10 Hz, preferably greater than 20 Hz.
The motion of the deposit device toward and away from the receiving surface is damped, preferably by friction or by a damping material associated with the support of the deposit device.
The apparatus of any of the foregoing is preferably constructed to mount a number of microscope slides or slide-like structures to serve as the receiving surface, and a control system is constructed and arranged to deposit drops of fluid in selected locations on the slides or slide-like structures, preferably the fluid source comprising a source of biological fluid.
Another broad aspect of the invention is a fluid deposit assembly mounted on a carrier for depositing minute drops of fluid at selected locations upon a receiving surface, comprising a deposit device having an exposed tip, preferably of diameter of 375 or 300 micron or less, constructed and arranged to carry and deposit drops of fluid upon the surface, stable lateral reference surfaces or surface portions exposed for engagement by the deposit device, the surfaces or surface portions constructed and arranged to prevent lateral displacement of the deposit device relative to the carrier when the deposit device is urged thereagainst and means for urging the deposit device against the reference surfaces or surface portions at least at the time that the deposit device approaches the receiving surface to deposit a fluid drop, the reference surfaces or surface portions and the means for urging cooperating to position the deposit tip in a precisely desired position as it contacts the receiving surface.
Preferably, for depositing fluid drops in a dense array of mutually isolated dots, the assembly comprises a fluid source for repeatedly providing a discrete drop of fluid on the tip of the deposit device, mechanism for moving the device relatively over an array of spaced apart deposit locations of a receiving surface, mechanism for repeatedly moving the device, relatively, toward and away from the receiving surfaces to deposit respective dots at respective deposit locations on the surface, preferably the fluid source being a mobile fluid storage device separate from the deposit device, which is generally movable over the array of deposit locations, the fluid storage device being constructed and arranged to resupply the deposit device at various locations with respect to the array.
In certain preferred embodiments of this aspect also, the deposit device is a slidable pin or pin-like structure constructed and arranged to dip into a volume of fluid carried by a mobile storage device, preferably the storage device being constructed to store a multiplicity of isolated fluid volumes, the apparatus constructed to move the supply device relative to the deposit device to select the fluid to be deposited, preferably the storage device being a 96 well plate or a plate having a multiple of 96 wells, and also preferably including at least one driven stage for moving a selected well of a mobile multiwell plate into registry with the deposit device under computer control for enabling motion of the deposit device to dip into and out of the preselected well to provide a drop of the selected fluid to the device.
In other preferred embodiments the mobile storage device is an annular ring as described above.
In embodiments in which the deposit device is a pin or pin-like structure, it is preferably positioned by engagement with a surface of revolution whose axis is disposed at a predetermined relationship to the receiving surface or substrate, preferably the surface of revolution being in the form of a supporting ledge that supports the device from moving in its assembly in the direction toward the receiving surface, but from which the device is free to lift in response to contact of the tip of the device with the receiving surface as the supporting ledge and device are together moved relatively toward the receiving surface for depositing a drop, preferably the surface of revolution having a surface of form substantially matching the form of the portion of the device disposed to engage it, preferably the surfaces being respectively conical, each preferably conforming to a portion of the surface of a right cone.
In certain embodiments a means for urging the deposit device against reference surfaces applies a lateral force or turning moment to the deposit device, preferably the force or turning moment being applied by a spring bearing eccentrically on the device or by a pushing member engaged with a remote end of the deposit device, one of the engaged end and pushing member comprising a surface set at an acute angle to an axis of the device, and the other of the surfaces comprising a convexly curved surface engaged upon the angled surface, preferably the convexly curved surface defined by a confined ball that bears against the inclined surface, preferably by being pushed by a weight.
In embodiments in which the deposit device is in the form of a pin or pin-like structure, a structure prevents rotation of the deposit device about its own axis, preferably the pin or pin-like structure confined in a complementary space that prevents its rotation about its own axis or a detent prevents rotation of the deposit device, preferably the detent comprising part of a coil spring which surrounds and is frictionally secured to the pin or pin-like structure, a protrusion of the spring engaging a stop surface that prevents the rotation, preferably the spring also providing axial compliance to the pin or pin-like structure.
Another broad aspect of the invention is a deposit apparatus comprising a multiplicity of deposit devices as described, mounted for motion together in response to a common actuator, preferably the deposit devices comprising deposit pins or pin-like structures.
Another broad aspect of the invention is an apparatus comprising a mobile fluid storage device separate from a deposit device and generally movable over an array of deposit locations, the fluid storage device being constructed and arranged to resupply the deposit device at various locations with respect to the array, in one case, preferably the mobile fluid storage device being constructed to store a multiplicity of isolated fluid volumes, the apparatus constructed to move the mobile storage device relative to the deposit device to select the fluid to be deposited, preferably the deposit device being a pin or pin-like structure constructed and arranged, under computer control, to dip into a selected volume of fluid carried by the mobile fluid storage device, preferably the mobile fluid storage device being a multiwell plate having 96 wells or multiples of 96 wells, or a spacing of 9 mm or a sub-multiple of 9 mm and preferably the apparatus including a driven stage for moving the fluid storage device into registry with the deposit device under computer control for enabling dipping of the deposit device into a preselected fluid volume; in another case preferably the mobile storage device is an annular ring that retains a supply of fluid by surface tension.
The invention also features the method of use of all the described apparatus in depositing fluid drops, especially the fluids mentioned in the specification.
Another broad aspect of the invention is a method of depositing a biological compound on a substrate or causing biological compounds to interact with another substance at a predetermined position on a substrate, including the step of depositing at least one of the compound or substance in a precisely determined localized spot relative to the substrate by mechanically lowering a compliant deposit device, preferably a compliant pin or pin-like structure, to which a drop of the compound or substance is held by surface tension, toward the substrate until the pin or pin-like structure contacts the substrate or a pre-applied compound on the substrate and thereafter mechanically lifting the deposit device away from the substrate under conditions in which the fluid drop transfers to the substrate or the pre-applied compound on the substrate, preferably the deposit device, when approaching the substrate, applying a force to the substrate of less than about 1 gram, preferably less than 0.5 grams, preferably about 0.3 grams and preferably the drop being less than 300 micron in diameter, preferably less than 200 or 100 micron in some cases preferably superposed drops of both a compound and another substance being successively deposited by the said technique.
Preferably in certain cases the fluid supply of the biological compound or substance to be deposited by the pin or pin-like structure is obtained by dipping the pin or pin-like structure in fluid, or the deposit device is supported above the substrate at the deposit location within a ring holding fluid by surface tension, and the pin is moved through the ring in the manner that a relatively small drop of the fluid supply is held by the end of the pin or pin-like structure by surface tension, preferably in both cases the pin providing a drop-carrying surface bound by a sharp rim that sizes the drop.
Preferably the fluid to be deposited is fluid selected from a group of fluids disposed in a multiwell plate, either a plate which moves across the substrate to be in proximity to the deposit pin or pin-like structure, or a plate visited by an annular supply ring.
Another broad aspect of the invention is a method of producing arrays of fluid dots comprising providing an array of compliant deposit devices according to any of the foregoing claims, the devices preferably being in the form of pin or pin-like structures, the devices having spacings corresponding to the well spacing of a 96 well plate, or a plate having a multiple of 96 wells or a spacing of 9 mm or a submultiple of 9 mm, according to a sampling plan, preferably either dipping mobile annular supply rings into wells of the plate or dipping the devices directly into wells of the plate with which the device is registered to provide fluid drops on the devices and transferring the drops to respective locations in substantially denser arrays on a receiving surface, preferably the drops being deposited on a microscope slide in a pattern of square arrays.
In the various methods, preferably drops of fluid are deposited under computer control, by moving at least one compliantly mounted pin or pin-like structure having a drop-supporting surface of diameter less than about 375 microns, preferably less than 300 microns, preferably less than 250 micron, to a selected position and depositing, with the pin or pin-like structure, a desired material.
In the various methods, preferably the receiving surface is fragile, or soft, preferably the receiving surface is porous or microporous or fibrous, preferably comprising nitrocellulose, nylon cellulose acetate or polyvinylidine fluoride or a gel, preferably the member defining the soft or fragile receiving surface being mounted on a rigid carrier member, either directly or upon an intermediate soft or resilient buffer member.
Preferably the method is employed to deposit a fluid selected from the group of biological fluids described in the specification, preferably the material being a biological probe or a chemical for reaction with biological material, a fluorescing material, an ink, dye, stain or marker, a photoactive material, or a varnish or an encapsulant or an etchant, or a cleaning or neutralizing agent.
Another broad aspect of the invention is the method of depositing a biological fluid with a pin or pin-like structure comprising supporting fluid within a ring by surface tension, and moving the pin or pin-like structure through the ring in the manner that a relatively small drop of the fluid is held by to the end of the pin by surface tension and deposited on a receiving surface.
Preferably an array of deposits formed by any of the described methods is microscopically examined with a wide area scanning microscope.
The invention also features an array product comprising deposited dots of fluid of diameter less than about 375 micron diameter, preferably less than 300 micron, preferably between 15 or 50 and 250 micron, in a dense array in a pattern corresponding to a function of the distribution of wells of a 96 well plate, preferably the dots being spaced from each other in the array less than three times their diameter, preferably less than twice, or about one and one half times the dot diameter the deposits preferably residing upon a glass microscope slide or on a fragile or soft surface, preferably a porous or microporous surface, the surface preferably comprising nitrocellulose, nylon, cellulose acetate, polyvinylidine fluoride or a gel, the fragile or soft surface preferably mounted on a rigid support directly or via an intermediate soft or resilient buffer member.